Anti-malaria compositions and methods

ABSTRACT

Described herein are multilayer films that include modified polypeptide epitopes from  Plasmodium falciparum , specifically a modified T* epitope. The multilayer films are capable of eliciting an immune response in a host upon administration to the host. The multilayer films can include at least one designed peptide that includes the modified T* polypeptide epitope from a  Plasmodium  protozoan.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application62/219,260 filed on Sep. 16, 2015, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

This invention was made with government support under AI091089 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE DISCLOSURE

The present disclosure relates to compositions and methods for theprevention of malaria infections, specifically multilayer filmcompositions containing antigenic epitopes.

BACKGROUND

Malaria is one of the most prevalent infections in tropical andsubtropical areas throughout the world. Malaria infections lead tosevere illnesses in hundreds of millions of individuals worldwide,leading to death in millions of individuals, primarily in developing andemerging countries every year. The widespread occurrence and elevatedincidence of malaria are a consequence of the increasing numbers ofdrug-resistant parasites and insecticide-resistant parasite vectors.Other factors include environmental and climatic changes, civildisturbances, and increased mobility of populations.

Malaria is caused by the mosquito-borne hematoprotozoan parasitesbelonging to the genus Plasmodium. Four species of Plasmodium protozoa(P. falciparum, P. vivax, P. ovale and P. malariae) are responsible forthe disease in man; many others cause disease in animals, such as P.yoelii and P. berghei in mice. P. falciparum accounts for the majorityof human infections and is the most lethal type, sometimes called“tropical malaria”. Malaria parasites have a life cycle consisting ofseveral stages. Each stage is able to induce specific immune responsesdirected against the corresponding occurring stage-specific antigens. Acurrent area of focus is development of vaccines that elicit immunityagainst the sporozoite stage pathogen. The sporozoite grows in thesaliva of infected mosquitoes and is transferred to the human during themosquito bite. The sporozoite travels thorough the blood stream to theliver where it enters hepatocytes and multiplies. Sporozoites arecovered with many copies of the circumsporozoite coat protein (CS).Antibodies that bind to CS proteins can neutralize the organism andprevent liver invasion, so agents that elicit potent and long lastinganti-CS responses are expected to be useful malaria vaccines.

Currently there are two vaccines in clinical trials that seek to preventmalaria infections via the CS neutralization mechanism. RTS,S is a viruslike particle vaccine that presents multiple copies of CS on avirus-like particle. It has been shown to protect both adults andchildren from infection but since efficacy is less than 50% its utilityis still a matter for debate. Sanaria Inc. has proposed the use ofkilled sporozoites as an effective vaccine but the method of productioninvolves the dissection of host mosquito saliva glands, a process thatis tedious and may not be scalable to practical quantities. Hence thereis a need for improved antigenic compositions that elicit immuneresponses which recognize and neutralize the malaria organism.

SUMMARY

In one aspect, an isolated peptide comprises the sequence of SEQ ID NO:5.

In another aspect, a composition comprises

a first multilayer film comprising a plurality of oppositely chargedpolyelectrolyte layers, wherein one of the polyelectrolyte layers in themultilayer film comprises a first antigenic polyelectrolyte,

wherein the first antigenic polyelectrolyte comprises a modifiedPlasmodium falciparum circumsporozoite T* epitope of SEQ ID NO: 5, and

wherein the polyelectrolytes in the multilayer film comprise apolycationic material or a polyanionic material having a molecularweight of greater than 1,000 and at least 5 charges per molecule.

In another embodiment, a method of eliciting an immune response in avertebrate organism comprising administering into the vertebrateorganism the multilayer film composition described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the epitopes of P. falciparum CS protein showing thelocations and sequences of the T1, B, and T* epitopes.

FIG. 2 shows results for sera collected on day 49 from C57BL/6J miceimmunized with T1BT* microparticle constructs tested in ELISA againstT1B peptide.

FIG. 3 shows sera from C57BL/6J mice immunized with T1BT* microparticleconstructs tested at 1:250, where plates were probed withisotype-specific detection antibodies.

FIG. 4 shows the results for spleen cells harvested on day 49 fromC57BL/6J mice immunized with T1BT* microparticle constructs andrestimulated with T1B peptide in IFNγ and IL-5 ELISPOT plates.

FIG. 5 shows the results for sera harvested on day 59 from C57BL/6J miceimmunized with T1BT* microparticle constructs and tested in ELISAagainst T1B peptide.

FIG. 6 shows spleen cells harvested on day 59 from C57BL/6J miceimmunized with T1BT* microparticle constructs and restimulated with T1Bpeptide in IFNγ and IL-5 ELISPOT plates.

FIG. 7 shows the results for from C57BL/6J mice immunized with T1BT*microparticle constructs and then challenged with PfPb on day 63 andsacrificed 40 hours later. Parasite burden in the livers was measured byqPCR.

FIG. 8 shows HPLC chromatograms at 214 nm of T1BT*-K20 peptide (SEQ IDNO: 10) after various storage conditions. A) Purified peptide is asingle peak. B) After room temperature in pH approximately 5 solutionfor 16 days. C) After room temperature in pH 7.4 solution for 2.7 days.D) After room temperature in pH 7.4 solution for 16 days. E) Afterstorage at −20° C. as a frozen solution for 4 years.

FIG. 9 shows HPLC chromatograms recorded at 280 nm of T1BT*-K20 peptide(SEQ ID NO: 10) mixture. A) After room temperature in approximately pH 5solution for 16 days. B) After treatment with dithiothreitol.

FIG. 10 shows HPLC chromatograms at 214 nm of T1BT*-K20 peptide (SEQ IDNO: 10) mixture. A) Freshly dissolved sample. B) After incubation atroom temperature in pH 7.4 solution for 5 days. C) After treatment withdithiothreitol

FIG. 11 shows HPLC chromatograms at 214 nm of T1BT*-K20 peptide (SEQ IDNO: 17) mixture. A) Freshly dissolved sample. B) After incubation atroom temperature in pH 7.4 solution for 5 days.

FIG. 12 shows C4 HPLC chromatograms at 214 nm (top) and 280 nm (bottom)for purified Pam3Cys-T1BT*-K20 peptide (SEQ ID NO: 13)

FIG. 13 shows electrospray mass spectrum for purified Pam3Cys-T1BT*-K20peptide (SEQ ID NO: 13)

The above-described and other features will be appreciated andunderstood by those skilled in the art from the following detaileddescription, drawings, and appended claims.

DETAILED DESCRIPTION

Disclosed herein are multilayer films comprising modified polypeptideepitopes from a Plasmodium protozoan, wherein the multilayer films arecapable of eliciting an immune response in a host upon administration tothe host. Specifically, the films comprise one or more Plasmodiumfalciparum circumsporozoite protein antigens, wherein thecircumsporozoite protein antigens include a modified T* epitope or amodified T1BT* epitope.

As used herein, the Plasmodium falciparum circumsporozoite proteinantigens are:

T1:  (SEQ ID NO: 1) DPNANPNVDPNANPNV B:  (SEQ ID NO: 2) NANPNANPNANPT*:  (SEQ ID NO: 3) EYLNKIQNSLSTEWSPCSVT T1BT*:  (SEQ ID NO: 4)DPNANPNVDPNANPNVNANPNANPNANPEYLNKIQNSLS TEWSPCSVT

In one aspect, a modified T* epitope is:

(SEQ ID NO: 5) EYLNKIQNSLSTEWSPSSVT,  or (SEQ ID NO: 6)EYLNKIQNSLSTEWSPASVT.

In a related aspect, a modified T1BT* epitope is:

(SEQ ID NO: 7) DPNANPNVDPNANPNVNANPNANPNANPEYLNKIQNSLSTEWSPSSV T,  or(SEQ ID NO: 8) DPNANPNVDPNANPNVNANPNANPNANPEYLNKIQNSLSTEWSPASV T.

During production of designed peptides for inclusion in multilayerfilms, there was a concern that interchain disulfide bonds might beformed during peptide synthesis or film production. The modified T*epitopes of SEQ ID Nos. 5 and 6 were designed to modify the unpaired Cysresidue in the wild-type T* epitope to provide a significant advantageduring the manufacturing process of designed peptides and multilayerfilms. When microparticles containing designed T1BT* peptides with themodified T* epitopes of SEQ ID Nos. 5 and 6 were tested in an animalmodel, it was found that while peptides containing SEQ ID NO: 5 eliciteda T-cell response specific for the wild-type T1B peptide (SEQ ID NO: 4),peptides containing SEQ ID NO: 6 failed to elicit a T-cell responsespecific for the wild-type T1B peptide. It was completely unexpectedthat the substitution of a Ser residue would be tolerated, while an Alasubstitution would not be tolerated.

Specifically, multilayer films comprise alternating layers of oppositelycharged polyelectrolytes, wherein one of the layers comprises a modifiedT* peptide, or a T1BT* peptide containing a modified T* epitope,specifically SEQ ID NO: 5 (modified T* epitope) or SEQ ID NO: 7(modified T1BT* epitope). Optionally, one or more of thepolyelectrolytes, specifically a polyelectrolyte comprising the modifiedT* or modified T1BT* peptide is a polypeptide. In certain embodiments,the multilayer films comprise multiple epitopes from a Plasmodiumprotozoan. For example, first and second Plasmodium protozoanpolypeptide epitopes can be attached to the same or differentpolyelectrolytes, and/or can be present in the same or differentmultilayer film.

In one aspect, the modified T* peptide, or a T1BT* peptide containing amodified T* epitope, is covalently linked to a polycationic material ora polyanionic material having a molecular weight of greater than 1,000and at least 5 charges per molecule. The polycationic or polyanionicmaterial provides sufficient charge for deposition of the modified T*peptide or T1BT* peptide containing a modified T* epitope into a layerof a multilayer film.

In another aspect, the modified T* peptide or T1BT* peptide containing amodified T* epitope is covalently linked to one or two surfaceadsorption regions at the C-terminus and/or the N-terminus of thepolypeptide, wherein at least one of the surface adsorption regionscomprises five or more, such as 10 to 20, negatively or positivelycharged amino acid residues. The surface adsorption regions providesufficient charge for deposition of the modified T* peptide or T1BT*peptide containing a modified T* epitope into a layer of a multilayerfilm. In one embodiment, the net charge per residue of the antigenicpolypeptide (including the T* epitope and the surface adsorptionregions) is greater than or equal to 0.1, 0.2, 0.3, 0.4, or 0.5 at pH7.0.

In one embodiment, a composition comprises a first multilayer filmcomprising a plurality of oppositely charged polyelectrolyte layers,wherein one of the polyelectrolyte layers in the multilayer filmcomprises a first antigenic polyelectrolyte, wherein the first antigenicpolyelectrolyte comprises a modified Plasmodium falciparumcircumsporozoite T* epitope, and wherein the polyelectrolytes in themultilayer film comprise a polycationic material or a polyanionicmaterial having a molecular weight of greater than 1,000 and at least 5charges per molecule. In another embodiment, a composition comprises afirst multilayer film comprising a plurality of oppositely chargedpolyelectrolyte layers, wherein one of the polyelectrolyte layers in themultilayer film comprises a first antigenic polyelectrolyte, wherein thefirst antigenic polyelectrolyte comprises a modified Plasmodiumfalciparum circumsporozoite T1BT* epitope, and wherein thepolyelectrolytes in the multilayer film comprise a polycationic materialor a polyanionic material having a molecular weight of greater than1,000 and at least 5 charges per molecule. In one aspect, the modifiedT* epitope has SEQ ID NO: 5. In another aspect, the modified T1BT*epitope has SEQ ID NO: 7.

In one embodiment, the first antigenic polyelectrolyte comprises two ofthe Plasmodium falciparum circumsporozoite epitopes, such as T1T*, BT*,in any order, wherein the T* epitope is a modified T* epitope. Theepitopes can be contiguous on the polypeptide chain, or spaced by aspacer region. Similarly, the epitopes can be at the N-terminus of thepolypeptide, the C-terminus of the polypeptide, or anywhere in between.In yet another embodiment, the first polyelectrolyte is a polypeptidecomprising all three of the Plasmodium falciparum circumsporozoite T1,B, and modified T* epitopes. The T1, B, and modified T* epitopes can bein a contiguous part of the polypeptide, or any or all of the epitopescan be separated by a spacer region.

In one aspect, the modified T* peptide, or a T1BT* peptide containing amodified T* epitope, in the multilayer film is covalently linked to apolycationic material or a polyanionic material having a molecularweight of greater than 1,000 and at least 5 charges per molecule. Thepolycationic or polyanionic material provides sufficient charge fordeposition of the modified T* peptide or T1BT* peptide containing amodified T* epitope into a layer of a multilayer film.

In one aspect, in order to facilitate deposition of the modified T* ormodified T1BT* epitope into a multilayer film, the peptide comprises oneor more highly charged surface adsorption regions, such as at theN-terminus, the C-terminus, or both. In one aspect, at least one of thesurface adsorption regions comprises five or more negatively orpositively charged amino acid residues. Peptides containing an antigenicpeptide and one or more surface adsorption regions are denoted herein asdesigned polypeptides (DP).

It is noted that when the first antigenic polyelectrolye is apolypeptide, the polypeptide contains sufficient charge for depositioninto a polypeptide multilayer film. In one embodiment, the net chargeper residue of the polypeptide is greater than or equal to 0.1, 0.2,0.3, 0.4 or 0.5 at pH 7.0, as explained herein.

In another embodiment, instead of the Plasmodium falciparumcircumsporozoite T1, B and modified T* epitopes being on the samepolyelectrolyte, two or three epitopes can be presented on separatepolyelectrolytes, and layered into the same multilayer film. In oneembodiment, the first multilayer film further comprises a secondantigenic polyelectrolyte comprising a Plasmodium falciparumcircumsporozoite T1, B, or modified T* epitope covalently linked to asecond polyelectrolyte, wherein the first and second antigenicpolyelectrolytes comprise different Plasmodium falciparumcircumsporozoite epitopes. In a further embodiment, the first multilayerfilm further comprises a third antigenic polyelectrolyte comprising aPlasmodium falciparum circumsporozoite T1, B, or modified T* epitopecovalently linked to a third polyelectrolyte, wherein the first, secondand third antigenic polyelectrolytes comprise different Plasmodiumfalciparum circumsporozoite epitopes. In one embodiment, the first,second and/or third polyelectrolyte is a polypeptide.

In one embodiment, a first, second and optionally third polyelectrolyteis presented in a separate multilayer film, such as two or threeindividual populations of coated cores, each population comprising adifferent multilayer film. Thus, in one embodiment, a compositioncomprises a first multilayer film as described above and a secondmultilayer film comprising a plurality of oppositely chargedpolyelectrolyte layers, wherein one of the layers in the secondmultilayer film comprises a second antigenic polyelectrolyte, whereinthe second antigenic polyelectrolyte comprises a Plasmodium falciparumcircumsporozoite T1, B or modified T* epitope covalently linked to asecond polyelectrolyte, wherein the first and second antigenicpolyelectrolytes comprise different Plasmodium falciparumcircumsporozoite epitopes. In a further embodiment, the compositionfurther comprises a third multilayer film comprising a plurality ofoppositely charged polyelectrolyte layers, wherein one of the layers inthe third multilayer film comprises a third antigenic polyelectrolyte,wherein the third antigenic polyelectrolyte comprises a Plasmodiumfalciparum circumsporozoite T1, B, or modified T* epitope covalentlylinked to a third polyelectrolyte, wherein the first, second and thirdantigenic polyelectrolytes comprise different Plasmodium falciparumcircumsporozoite epitopes. In certain embodiments, the first, second andor third polyelectrolyte is a polypeptide. In some embodiments, thefirst, second and third multilayer films are layered onto separate coreparticles, such that a composition comprises two or three distinctpopulations of particles.

In certain embodiments, the multilayer films further comprise atoll-like receptor ligand. As used herein, toll-like receptor ligands,or TLR ligands, are molecules that bind to TLRs and either activate orrepress TLR receptors. Activation of TLR signaling through recognitionof pathogen-associated molecular patterns (PAMPs) and mimics leads tothe transcriptional activation of genes encoding pro-inflammatorycytokines, chemokines and co-stimulatory molecules, which can controlthe activation of the antigen-specific adaptive immune response. TLRshave been pursued as potential therapeutic targets for variousinflammatory diseases and cancer. Following activation, TLRs induce theexpression of a number of protein families, including inflammatorycytokines, type I interferons, and chemokines. TLR receptor ligands canfunction as adjuvants for the immune response.

Exemplary TLR ligands include a TLR1 ligand, a TLR2 ligand, a TLR3ligand, a TLR4 ligand, a TLR5 ligand, a TLR6 ligand, a TLR 7 ligand, aTLR8 ligand, a TLR9 ligand and combinations thereof.

Exemplary TLR1 ligands include bacterial lipopeptides. Exemplary TLR2ligands include lipopeptides such as Pam3Cys([N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteine]) and Pam2Cys([S-[2,3-bis(palmitoyloxy)propyl]cysteine]). Exemplary TLR6 ligands arediacyl lipopeptides. TLR1 and TLR6 require heterodimerization with TLR2to recognize ligands. TLR1/2 are activated by triacyl lipoprotein (or alipopeptide, such as a Pam3Cys peptide), whereas TLR6/2 are activated bydiacyl lipoproteins (e g., Pam2Cys), although there may be somecross-recognition.

An exemplary TLR3 ligand is Poly(I:C). Exemplary TLR4 ligands arelipopolysaccharide (LPS) and monophospholipid A (MPLA). An exemplaryTLR5 ligand is flagellin. An exemplary TLR7 ligand is imiquimod. Anexemplary TLR8 ligand is single-stranded RNA. An exemplary TLR9 ligandis unmethylated CpG Oligodeoxynucleotide DNA.

In one embodiment, the first, second, or third antigenicpolyelectrolyte, e.g., an antigenic polypeptide, has a TLR ligandcovalently attached thereto. For example, Pam3Cys can be covalentlycoupled to a polypeptide chain by standard polypeptide synthesischemistry.

In another embodiment, a substrate such as a template core has depositedthereon a TLR ligand prior to deposition of polyelectrolyte layers. Inanother embodiment, a TLR ligand is co-deposited with one or morepolyelectrolyte layers during assembly of the multilayer film.

In one embodiment, the multilayer film is deposited on a core particle,such as a core nanoparticle or a core microparticle. Exemplary coresinclude CaCO₃ nanoparticles and microparticles, latex particles, andiron particles. Particle sizes on the order of 5 nanometers (nm) to 500micrometers (μm) in diameter are useful. Particles having diameters of0.05-20 μm are preferred for vaccine purposes. Particles of approximatediameter 1-10 μm are particularly useful as vaccines Particles made ofother materials can also be used as cores provided that they arebiocompatible, have controllable size distribution, and have sufficientsurface charge (either positive or negative) to bind polyelectrolytepeptides. Examples include nanoparticles and microparticles made ofmaterials such as polylactic acid (PLA), polylactic acid glycolic acidcopolymer (PLGA), polyethylene glycol (PEG), chitosan, hyaluronic acid,gelatin, or combinations thereof. Core particles could also be made ofmaterials that are believed to be inappropriate for human use providedthat they can be dissolved and separated from the multilayer filmfollowing film fabrication. Examples of the template core substancesinclude organic polymers such as latex or inorganic materials such assilica.

Polyelectrolyte multilayer films are thin films (e.g., a few nanometersto micrometers thick) composed of alternating layers of oppositelycharged polyelectrolytes. Such films can be formed by layer-by-layerassembly on a suitable substrate. In electrostatic layer-by-layerself-assembly (“LBL”), the physical basis of association ofpolyelectrolytes is electrostatic attraction. Film buildup is possiblebecause the sign of the surface charge density of the film reverses ondeposition of successive layers. The generality and relative simplicityof the LBL film process permits the deposition of many different typesof polyelectrolyte onto many different types of surface. Polypeptidemultilayer films are a subset of polyelectrolyte multilayer films,comprising at least one layer comprising a charged polypeptide, hereinreferred to as a designed polypeptide (DP). A key advantage ofpolypeptide multilayer films over films made from other polymers istheir biocompatibility.

LBL films can also be used for encapsulation of other materials.Applications of polypeptide films and microcapsules include, forexample, nano-reactors, biosensors, artificial cells, and drug deliveryvehicles.

The term “polyelectrolyte” includes polycationic and polyanionicmaterials having a molecular weight of greater than 1,000 and at least 5charges per molecule. Suitable polycationic materials include, forexample, polypeptides and polyamines. Polyamines include, for example, apolypeptide such as poly-L-lysine (PLL) or poly-L-ornithine, polyvinylamine, poly(aminostyrene), poly(aminoacrylate), poly (N-methylaminoacrylate), poly (N-ethylaminoacrylate), poly(N,N-dimethylaminoacrylate), poly(N,N-diethylaminoacrylate), poly(aminomethacrylate),poly(N-methyl amino-methacrylate), poly(N-ethyl aminomethacrylate),poly(N,N-dimethyl aminomethacrylate), poly(N,N-diethylaminomethacrylate), poly(ethyleneimine), poly (diallyl dimethylammoniumchloride), poly(N,N,N-trimethylaminoacrylate chloride),poly(methyacrylamidopropyltrimethyl ammonium chloride), chitosan andcombinations comprising one or more of the foregoing polycationicmaterials. Suitable polyanionic materials include, for example, apolypeptide such as poly-L-glutamic acid (PGA) and poly-L-aspartic acid,a nucleic acid oligomer such as DNA and RNA, alginate, carrageenan,furcellaran, pectin, xanthan, hyaluronic acid, heparin, heparan sulfate,chondroitin sulfate, dermatan sulfate, dextran sulfate,poly(meth)acrylic acid, oxidized cellulose, carboxymethyl cellulose,acidic polysaccharides, and croscarmelose, synthetic polymers andcopolymers containing pendant carboxyl groups, and combinationscomprising one or more of the foregoing polyanionic materials. In oneembodiment, the Plasmodium protozoan epitope and the polyelectrolytehave the same sign of charge. In another embodiment, the Plasmodiumprotozoan epitope and the polyelectrolyte have the opposite sign ofcharge.

In one embodiment, one or more polyelectrolyte layers of the film,optionally including the polyelectrolyte comprising the Plasmodiumprotozoan epitope, is a designed polypeptide. In one embodiment, thedesign principles for polypeptides suitable for electrostaticlayer-by-layer deposition are elucidated in U.S. Patent Publication No.2005/0069950, incorporated herein by reference for its teaching ofpolypeptide multilayer films. Briefly, the primary design concerns arethe length and charge of the polypeptide. Electrostatics is the mostimportant design concern because it is the basis of LBL. Withoutsuitable charge properties, a polypeptide may not be substantiallysoluble in aqueous solution at pH 4 to 10 and cannot readily be used forthe fabrication of a multilayer film by LBL. Other design concernsinclude the physical structure of the polypeptides, the physicalstability of the films formed from the polypeptides, and thebiocompatibility and bioactivity of the films and the constituentpolypeptides.

A designed polypeptide means a polypeptide that has sufficient chargefor stable binding to an oppositely charged surface, that is, apolypeptide that can be deposited into a layer of a multilayer filmwherein the driving force for film formation is electrostaticattraction. A short stable film is a film that once formed, retains morethan half its components after incubation in PBS at 37° C. for 24 hours.In specific embodiments, a designed polypeptide is at least 15 aminoacids in length and the magnitude of the net charge per residue of thepolypeptide is greater than or equal to 0.1, 0.2, 0.3, 0.4 or 0.5 at pH7.0. Positively-charged (basic) naturally-occurring amino acids at pH7.0 are arginine (Arg), histidine (His), ornithine (Orn), and lysine(Lys). Negatively-charged (acidic) naturally-occurring amino acidresidues at pH 7.0 are glutamic acid (Glu) and aspartic acid (Asp). Amixture of amino acid residues of opposite charge can be employed solong as the overall net ratio of charge meets the specified criteria. Inone embodiment, a designed polypeptide is not a homopolymer. In anotherembodiment, a designed polypeptide is unbranched.

The electrostatic attraction between oppositely charged polyelectrolytesis usually sufficient to produce a film that is stable under ambientconditions, for example at neutral pH and body temperature. However,film stability can be increased by engineering covalent bonds betweenthe layers after the film is formed. This cross-linking process confersadditional stability upon the film and can enable it to withstand morestringent conditions such higher temperatures or large changes in pH.Examples of covalent bonds useful for cross-linking include disulfidesbonds, thioether bonds, amide bonds, and others. For films comprised ofpolypeptides, chemistries that produce amide bonds are particularlyuseful. In the presence of appropriate coupling reagents, acidic aminoacids (those with side chains containing carboxylic acid groups such asaspartic acid and glutamic acid) will react with amino acids whose sidechains contain amine groups (such as lysine and ornithine) to form amidebonds. Amide bonds are more stable than disulfide bonds under biologicalconditions and amide bonds will not undergo exchange reactions. Manyreagents can be used to activate polypeptide side chains for amidebonding. Carbodiimide reagents, such as the water soluble1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) will react withaspartic acid or glutamic acid at slightly acidic pH, forming anintermediate product that will react irreversibly with an amine toproduce an amide bond. Additives such as N-hydroxysuccinimide (NHS) areoften added to the reaction to accelerate the rate and efficiency ofamide formation. After cross-linking the soluble reagents and byproductsare removed from the nanoparticles or microparticles by, for example,centrifugation and aspiration. Alternatively, soluble reagents can beremoved by filtration of the particles, for example, by tangential flowfiltration (TFF). Examples of other coupling reagents includediisopropylcarbodiimide, HBTU, HATU, HCTU, TBTU, and PyBOP. Examples ofother additives include sulfo-N-hydroxysuccinimide (sulfo-NHS),1-hydroxbenzotriazole, and 1-hydroxy-7-aza-benzotriazole. The extent ofamide cross-linking can be controlled by modulating the stoichiometry ofthe coupling reagents, the time of reaction, or the temperature of thereaction, and can be monitored by techniques such as Fouriertransform-infrared spectroscopy (FT-IR).

Covalently cross-linked LBL films have desirable properties such asincreased stability. Greater stability allows for more stringentconditions to be used during nanoparticle, microparticle, nanocapsule,or microcapsule fabrication. Examples of stringent conditions includehigh temperatures, low temperatures, cryogenic temperatures, highcentrifugation speeds, high salt buffers, high pH buffers, low pHbuffers, filtration, and long term storage. In one aspect, at least twopolyelectrolyte layers of the multilayer film, other than the layercontaining the first antigenic polyelectrolyte, are covalentlycross-linked. The covalent cross-link bonds are, for example, amidebonds involving amino acid side chain functional groups.

A method of making a polyelectrolyte multilayer film comprisesdepositing a plurality of layers of oppositely charged chemical specieson a substrate. In one embodiment, at least one layer comprises adesigned polypeptide. Successively deposited polyelectrolytes will haveopposite net charges. In one embodiment, deposition of a polyelectrolytecomprises exposing the substrate to an aqueous solution comprising apolyelectrolyte at a pH at which it has a suitable net charge for LBL.In other embodiments, the deposition of a polyelectrolyte on thesubstrate is achieved by sequential spraying of solutions of oppositelycharged polypeptides. In yet other embodiments, deposition on thesubstrate is by simultaneous spraying of solutions of oppositely chargedpolyelectrolytes.

In the LBL method of forming a multilayer film, the opposing charges ofthe adjacent layers provide the driving force for assembly. It is notcritical that polyelectrolytes in opposing layers have the same netlinear charge density, only that opposing layers have opposite netcharges. One standard film assembly procedure by deposition includesforming aqueous solutions of the polyelectrolytes at a pH at which theyare ionized (i.e., pH 4-10), providing a substrate bearing a surfacecharge, and alternating immersion of the substrate into the chargedpolyelectrolyte solutions. The substrate is optionally washed in betweendeposition of alternating layers to remove unbound polyelectrolyte.

The concentration of polyelectrolyte suitable for deposition of thepolyelectrolyte can readily be determined by one of ordinary skill inthe art. An exemplary concentration is 0.1 to 10 mg/mL.

In addition, the number of layers required to form a stablepolyelectrolyte multilayer film will depend on the polyelectrolytes inthe film. For films comprising only low molecular weight polypeptidelayers, a film will typically have two or more bilayers of oppositelycharged polypeptides. Studies have shown that polyelectrolyte films aredynamic. The polyelectrolytes contained within a film can migratebetween layers and can exchange with soluble polyelectrolytes of likecharge when suspended in a polyelectrolyte solution. Moreoverpolyelectrolyte films can disassemble or dissolve in response to achange in environment such as temperature, pH, ionic strength, oroxidation potential of the suspension buffer. Thus some polyelectrolytesand particularly peptide polyelectrolytes exhibit transient stability.The stability of peptide polyelectrolyte films can be monitored bysuspending the films in a suitable buffer under controlled conditionsfor a fixed period of time, and then measuring the amounts of thepeptides within the film with a suitable assay such as amino acidanalysis, HPLC assay, or fluorescence assay. Peptide polyelectrolytefilms are most stable under conditions that are relevant to theirstorage and usage as vaccines, for example in neutral buffers and atambient temperatures such as 4° C. to 37° C. Under these conditionsstable peptide polyelectrolyte films will retain most of their componentpeptides for at least 24 hours and often up to 14 days and beyond.

In one embodiment, a designed polypeptide comprises one or more surfaceadsorption regions covalently linked to one or more Plasmodium protozoanepitopes. As used herein, a surface adsorption region is a chargedregion of a designed polypeptide that advantageously provides sufficientcharge so that a peptide containing an epitope from a Plasmodiumprotozoan, for example, can be deposited into a multilayer film. The oneor more surface adsorption regions and the one or more Plasmodiumprotozoan epitopes can have the same or opposite polarity. In anotherembodiment, the solubility of the designed polypeptide at pH 4 to 10 isgreater than or equal to about 0.1 mg/mL. In another embodiment, thesolubility of the designed polypeptide at pH 4 to 10 is greater than orequal to about 1 mg/mL. The solubility is a practical limitation tofacilitate deposition of the polypeptides from aqueous solution.

An exemplary surface adsorption region comprises 20 consecutive lysineresidues (K₂₀ or K₂₀Y). When the Plasmodium protozoan epitope is themodified T1BT* epitope of SEQ ID NO: 7, for example, it is preferredthat the surface adsorption region(s) include 5 to 20 positively chargedamino acid residues, or 5 to 20 negatively charged amino acid residues.

In one embodiment, a designed polypeptide comprises a single antigenicPlasmodium protozoan epitope flanked by two surface adsorption regions,an N-terminal surface adsorption region and a C-terminal surfaceadsorption region. In another embodiment, a designed polypeptidecomprises a single antigenic Plasmodium protozoan epitope flanked by onesurface adsorption region linked to the N-terminus of the Plasmodiumprotozoan epitope. In another embodiment, a designed polypeptidecomprises a single antigenic Plasmodium protozoan epitope flanked by onesurface adsorption region linked to the C-terminus of the Plasmodiumprotozoan epitope.

Each of the independent regions (e.g., Plasmodium protozoan epitopes andsurface adsorption regions) of the designed polypeptide can besynthesized separately by solution phase peptide synthesis, solid phasepeptide synthesis, or genetic engineering of a suitable host organism. Acombination of solution phase and solid phase methods can be used tosynthesize relatively long peptides and even small proteins. Peptidesynthesis companies have the expertise and experience to synthesizedifficult peptides on a fee-for-service basis. The syntheses areperformed under good manufacturing practices (GMP) condition and atscales suitable for clinical trials and commercial drug launch.

Alternatively, the various independent regions can be synthesizedtogether as a single polypeptide chain by solution-phase peptidesynthesis, solid phase peptide synthesis or genetic engineering of asuitable host organism. The choice of approach in any particular casewill be a matter of convenience or economics.

If the various Plasmodium protozoan epitopes and surface adsorptionregions are synthesized separately, once purified, for example, by ionexchange chromatography or by high performance liquid chromatography,they can be joined by peptide bond synthesis. That is, the N-terminus ofthe surface adsorption region and the C-terminus of one or more of thePlasmodium protozoan epitopes are covalently joined to produce thedesigned polypeptide. Alternatively, the C-terminus of the surfaceadsorption region and the N-terminus of the Plasmodium protozoan epitopeare covalently joined to produce the designed polypeptide. Theindividual fragments can be synthesized by solid phase methods andobtained as fully protected, fully unprotected, or partially protectedsegments. The segments can be covalently joined in a solution phasereaction or solid phase reaction. If one polypeptide fragment contains acysteine as its N-terminal residue and the other polypeptide fragmentcontains a thioester or a thioester precursor at its C-terminal residuethe two fragments will couple spontaneously in solution by a specificreaction commonly known to those skilled in the art as native cysteineligation. Native cysteine ligation is a particularly attractive optionfor designed peptide synthesis because it can be performed with fullydeprotected or partially protected peptide fragments in aqueous solutionand at dilute concentrations.

In one embodiment, the Plasmodium protozoan epitopes and/or surfaceadsorption regions are joined by peptidic or non-peptidic linkages asdescribed in U.S. Pat. No. 7,723,294, incorporated herein by referencefor its teaching of the use of non-peptidic linkages to join segments ofpolypeptides for use in multilayer films. Suitable non-peptidic linkersinclude, for example, alkyl linkers such as —NH—(CH₂)_(s)—C(O)—, whereins=2-20. Alkyl linkers are optionally substituted by a non-stericallyhindering group such as lower alkyl (e.g., C₁-C₆), lower acyl, halogen(e.g., Cl, Br), CN, NH₂, phenyl, and the like. Another exemplarynon-peptidic linker is a polyethylene glycol linker such as—NH—(CH₂—CH₂—O)_(n), —C(O)— wherein n is such that the linker has amolecular weight of about 100 to about 5000 Da. Many of the linkersdescribed herein are available from commercial vendors in a formsuitable for use in solid phase peptide synthesis.

In one embodiment, one or more of the polypeptide epitopes from aPlasmodium protozoan is covalently attached to one or more of thepolyelectrolyes, such as a polypeptide or other polyelectrolyte, throughcovalent bonds. Examples of suitable covalent bonds include amides,esters, ethers, thioethers, and disulfides. One skilled in the art cantake advantage of a range of functional groups found within the epitopepeptide to engineer a bond to a suitable electrolyte. For instance, acarboxylic acid in the epitope peptide can be found either at theC-terminal or on the side chain of amino acids aspartic acid or glutamicacid. Carboxylic acids can be activated with suitable peptide couplingreagents such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)for reaction with primary or secondary amines that are found in peptidepolyelectrolytes such as poly-L-lysine. The resulting amide bond isstable under ambient conditions. Conversely, the acid groups in apeptide polyelectrolyte can be activated with EDC for reaction withamine groups in the epitope peptide. Useful amine groups can be found atthe epitope peptide's N-terminal or on the side chain of lysineresidues.

Epitope peptides can also be attached to polyelectrolytes via thioetherbonds. Synthetic epitope peptides can be synthesized with appropriateelectrophiles such as haloacetyl groups which react specifically withsulfhydryls. For instance, an epitope peptide containing a chloroacetylat its N-terminal will form a stable bond to sulfhydryl bearingpolyelectrolytes.

Epitope peptides can also be attached covalently to polyelectrolytesthrough bifunctional linker molecules. Bifunctional linkers usuallycontain two electrophilic groups that can react with nucleophilespresent on either the epitope peptide or the polyelectrolyte molecule.Two classes of linker molecules are sold commercially, homobifunctionallinkers and heterobifunctional linkers. Homobifunctional linkers containtwo copies of an electrophilic group joined by a nonreactive spacer.Often the electophiles are active esters, such as N-hydroxysuccinimide(NHS) esters or sulfo-N-hyrdoxysuccinimide esters (sulfo-NHS) whichreact with nucleophilic amines. Examples of homobifunctional NHS estersinclude bis(sulfosuccinimidyl) suberate, disuccinimidyl glutarate,dithiobis(succinimidyl) propionate, disuccinimidyl suberate,disuccinimidyl tartrate. Sometimes the electophiles are aldehyde groupsthat form imides with nucleophilic amines on the epitope andpolyelectrolyte molecules. The imide bonds are transiently stable butcan be converted to stable structures with reducing agents such assodium borohydride or catalytic hydrogenation. The most commonly usedhomobifunctional aldehyde linker is glutaraldehyde.

Other commonly used homobifunctional linkers contain electrophiles thatreact specifically with nucleophilic thiols, which can be used to linkcysteine containing epitope peptides to sulfhydryl containingpolyelectrolytes as described above. Examples of sulfhydryl specifichomobifunctional linkers include 1,4-bismaleimidobutane, 1,4bismaleimidyl-2,3-dihydroxybutane, vbismaleimidohexane,bis-maleimidoethane, 1,4-di-[3′-(2′-pyridyldithio)-propionamido]butane,dithio-bismaleimidoethane, 1,6-hexane-bis-vinylsulfone.

While designed polypeptides containing free cysteine residues are usefulfor covalent linking to other molecules, the free sulfhydral group canoxidize in a variety of ways that can be problematic. For example, twofree cysteine containing peptides can undergo oxidative coupling andbecome covalently linked via a disulfide bond. When the free cysteinepeptides are identical, the product is referred to a symmetricaldisulfide or a disulfide dimer. Disulfide dimerization can occur undermild conditions, for example under ambient temperatures or even duringcold storage. All that is required is brief exposure to a mild oxidantsuch as air oxygen or another mild oxidant. Moreover dimerization canoccur when the free cysteine peptide is stored as a solid, for exampleas a dry powder, or in solution form, for example as a 1 mg/mL solutionin water at neutral pH. In cases where the free cysteine peptides areprone to self-associate, essentially all of the monomeric free cysteinepeptide can be converted to dimer. Even when careful steps are taken toexclude exposure to oxidants such as, for example, working in inert gasatmospheres or degassing of solvents and buffers, trace amounts orunwanted disulfide product can form.

Disulfide dimerization is a concern for vaccine compositions for anumber of reasons. First, if a free cysteine peptide is part of a drugsubstance, the disulfide dimer would be considered an impurity and wouldrequire frequent monitoring, quantitation, and if present in sufficientamounts, removal and/or characterization for possible toxicities.Secondly, since disulfide dimers can accumulate over time, a freecysteine peptide drug substance is likely to have a shorter shelf lifethan peptides lacking free cysteines. Thirdly, if the cysteine is partof or near an important antibody or T-cell epitope, dimerization mayobscure that epitope thus weakening the desired immune response. Andfinally, dimerization may create new antibody epitopes that areirrelevant to protection and thus dilute the desired immune response.Clearly, it is preferable to avoid using free cysteine peptides in drugsubstances unless the cysteine serves an important function, forexample, if it is a critical component of an antibody or T-cell epitope,or will be used for conjugation or linking as described above.

In Example 5, a free cysteine containing T1BT*-K20 designed peptide (SEQID NO: 10) was subjected to various storage conditions and the purity ofthe peptide was monitored chromatographically. All storage conditionsgave rise to new species that can be separated from the startingmonomeric free cysteine peptide. This new peak is likely the result ofcysteine oxidation, presumably to the symmetrical disulfide dimer, aconclusion that is supported by the observation that treatment with thereducing agent dithiothreitol (DTT) causes the new peak to revert backto the original free cysteine peptide. One would predict that thisphenomenon likely would occur in all T1BT* peptides that contain a freecysteine. Thus there is a need to control or prevent this undesired sidereaction from occurring.

Epitopes that elicit protective immune responses can be identified in anumber of ways. Putative antibody epitopes can be identified by testingimmune sera or monoclonal antibodies against subunits, deletion mutants,or point mutants of pathogen proteins in assays such as ELISA. PutativeT-cell epitopes can be identified by testing immune peripheral bloodmononuclear cells against overlapping peptides spanning the length ofpathogen proteins in assays such as ELISPOT. In each case, the use ofmultiple overlapping and partially redundant subunit analytes allows oneskilled in the art to identify the putative epitope that is recognizedby the protective immune component, be it an antibody or a T-cell. Theputative protective epitopes can then be validated by immunizing naïveanimals with the defined epitope(s) and challenging with the pathogen ofinterest to determine whether the epitope-restricted immune responseprotects the host from infection or pathology.

After an antibody or T-cell epitope has been identified, it may bepossible to substitute one or more of the individual amino acid residueswithout loss of function. Unfortunately it is difficult to predict whichresidues can be substituted, and which other residues can serve assuitable replacements. Thus new analogs with potentially allowedsubstitute residues must be prepared and tested in animal models or insurrogate in vitro assays that are predictive of the desired in vivoresult. One can prepare a large number of peptide analogs using peptidearray synthesis technologies but screening these in animal models may betime and cost prohibitive. Under such resource restrictions one canreduce the number of analogs to prepare and screen by limitingsubstitutions to those residues of similar size and/or functionality.Examples of amino acid residue pairs that have similar size andfunctionality, and are good candidates for substitution are:serine/threonine (Ser/Thr), isoleucine/valine (Ile/Val),phenylalanine/tyrosine (Phe/Tyr), isoleucine/leucine (Ile/Leu),asparagine/glutamine (Asn/Gln), asparagine/aspartic acid (Asn/Asp),glutamine/glutamic acid (Gln/Glu), aspartic acid/glutamic acid(Asp/Glu), and arginine/lysine (Arg/Lys). Glycine and proline arestructurally distinct from the other natural amino acids so it would notbe surprising to find no allowable substitutions for those residues.

By virtue of its high reactivity, hydrogen bonding properties, andtendency to oxidize, cysteine is unique amongst the natural amino acids.While cysteine contains the same number of heavy (non-hydrogen) atoms asserine, its side chain sulfhydral group is much more acidic thanserine's hydroxyl group, and is partially ionized at neutral pH.Therefore, the best way to identify a substitute for a cysteine residuewithin a peptide epitope is it to make several sensible analogs and testthose in a predictive assay, such as a mouse immunization model.

The T* epitope in the malaria CS protein has been found to be animportant component of peptide based candidate malaria vaccines. Itcontains a cysteine residue (Cys334) that in the folded CS protein isdisulfide bonded to a residue (Cys369) which lies outside of the T*region. As a T-cell epitope, immune compositions containing T* are takenup by antigen presenting cells, processed into shorter linear peptidesegments, and bound to major histocompatibility complex (MHC) moleculesin a linear conformation. The peptide:MHC complexes then translocate tothe cell surface and present the peptide to T-cells that recognize thepeptide:MHC complex via the T-cell receptor (TCR). In the case of the T*epitope, intracellular processing by the antigen presenting cell willreduce the native disulfide bond leaving Cys334 as a free cysteine. Intheory it should be possible to replace Cys334 with another residueprovided that interactions critical for MHC binding and recognition byT-cells are preserved.

The synthesis of designed peptides that contain T* cysteinesubstitutions is described in Example 1 and their fabrication intomalaria vaccine microparticles is described in Example 2. Designedpeptides Pam3Cys-T1BT*-K20 (Cys→Ser, SEQ ID NO: 13) andPam3Cys-T1BT*-K20 (Cys→Ala, SEQ ID NO: 14) contain serine and alaninesubstitutions for Cys334, respectively. Microparticle vaccinescontaining these designed peptides as well as microparticles thatcontain the native free cysteine designed peptide Pam3Cys-T1BT*-K20 (SEQID NO: 11) were used to immunize mice as described in Example 3 andExample 4. Following immunizations the animals were challenged withmosquitos infected with a hybrid form of the mouse malaria organismPlasmodium bergheii (Pb) that has been genetically engineered to expressthe T1B repeat elements of the circumsporozoite (CS) coat protein fromthe human malaria organism Plasmodium falciparum (Pf). The PfPbtransgenic organism enables vaccines targeted against the Pf CS proteinto be tested in a challenge experiment in mice. In the experimentdescribed in Example 3, all mouse cohorts responded to immunizationswith strong anti-T1B polyclonal antibody responses (FIG. 5). This wasthe expected result as the various vaccine batches were similar in allrespects except for base layer cross-linking and the residue at position334 in the T* epitope. Closer inspection of the data reveals that theserine replacement (SEQ ID NO: 13) elicited mouse sera with anti-T1Btiters equal to the cysteine containing sequence (SEQ ID NO: 11) whilethe alanine replacement (SEQ ID NO: 14) elicited lower titer anti-T1Bsera. This result was unexpected as the substitution occurs in the T*region yet it appears to have an effect on antibody responses to the T1Bepitopes. Thus, the T* epitope appears to affect the magnitude of theT1B specific antibody response, and non-optimal replacements for thenative cysteine reduce the magnitude of the response.

In Example 4, T-cells were collected from mice immunized with thevaccine microparticle constructs with modified T* epitopes. When testedin T1B peptide specific T-cell assay, only the constructs with thenative cysteine residue or the replacement serine residue elicitedrobust responses. Cells from mice treated with the alanine substitutionconstructs yielded poor T-cell responses (FIG. 6). Moreover, when theanimals were subjected to challenge with the PbPf hybrid organism,animals immunized with microparticles containing the serine mutantshowed increased levels of protection relative to mice immunized withthe alanine constructs (FIG. 7). Without being held to theory, it isbelieved that the serine mutant of the T* peptide preserves importantmolecular interactions with either the MHC presentation molecule or theT-cell receptor, or both, while the alanine mutant is lacking in one ormore interactions. The functional result of the missing interactions isa weaker T-cell specific response and a lower level of protectionagainst the live PfPb organism.

Through the practice of protein crystallography, much has been learnedabout the MHC-antigen peptide-TCR interaction system. However, accurateprediction of MHC binding peptides from a protein primary sequence hasnot yet been reduced to an exact science. Thus T-cell epitopes stillneed to be identified and validated by empirical methods. Likewise,acceptable modifications to an identified T-cell epitope need be testedempirically. An epitope with a single point substitution may retain fullimmunological activity, or show reduced immunological activity,abolished immunological activity, or increased immunological activitycompared to the wild-type native sequence. It is also possible that anepitope with a single point substitution may exhibit activityqualitatively different from the wild-type sequence, such as alteringthe induced T-cell response between Th1 and Th2 phenotypes. Whilesuitable substitutions can be predicted, they must be tested andvalidated by empirical methods.

Further disclosed herein is an immunogenic composition, said immunogeniccomposition comprising a multilayer film comprising two or more layersof polyelectrolytes, wherein adjacent layers comprise oppositely chargedpolyelectrolytes, wherein one layer comprises a Plasmodium protozoanepitope. The immunogenic composition optionally further comprises one ormore layers comprising a designed polypeptide.

In one embodiment, an immunogenic composition further comprises a secondPlasmodium protozoan epitope in addition to the modified T* or modifiedT1BT* epitope, either on the same or different designed polypeptides. Inone embodiment, the immunogenic composition comprises a plurality ofunique antigenic polyelectrolytes. In another embodiment, theimmunogenic composition comprises a plurality of immunogenicpolyelectrolytes comprising multiple Plasmodium protozoan epitopeswithin each polyelectrolyte. An advantage of these immunogeniccompositions is that multiple antigenic determinants or multipleconformations of a single linear antigenic determinant can be present ina single synthetic vaccine particle. Such compositions with multipleantigenic determinants can potentially yield antibodies against multipleepitopes, increasing the odds that at least some of the antibodiesgenerated by the immune system of the organism will neutralize thepathogen or target specific antigens on cancer cells, for example.

The immunogenicity of an immunogenic composition may be enhanced in anumber of ways. In one embodiment, the multilayer film optionallycomprises one or more additional immunomodulatory bioactive molecules.Although not necessary, the one or more additional immunomodulatorybioactive molecules will typically comprise one or more additionalantigenic determinants. Suitable additional immunomodulatory bioactivemolecules include, for example, a drug, a protein, an oligonucleotide, anucleic acid, a lipid, a phospholipid, a carbohydrate, a polysaccharide,a lipopolysaccharide, a low molecular weight immune stimulatorymolecule, or a combination comprising one or more of the foregoingbioactive molecules. Other types of additional immune enhancers includea functional membrane fragment, a membrane structure, a virus, apathogen, a cell, an aggregate of cells, an organelle, or a combinationcomprising one or more of the foregoing bioactive structures.

In one embodiment, the multilayer film/immunogenic composition evokes aresponse from the immune system to a pathogen. In one embodiment, avaccine composition comprises an immunogenic composition in combinationwith a pharmaceutically acceptable carrier. Thus a method of vaccinationagainst a pathogenic disease comprises the administering to a subject inneed of vaccination an effective amount of the immunogenic composition.

Pharmaceutically acceptable carriers include, but are not limited to,large, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, inactive virus particles, and the like.Pharmaceutically acceptable salts can also be used in the composition,for example, mineral salts such as hydrochlorides, hydrobromides,phosphates, or sulfates, as well as the salts of organic acids such asacetates, proprionates, malonates, or benzoates. The composition canalso contain liquids, such as water, saline, glycerol, and ethanol, aswell as substances such as wetting agents, emulsifying agents, or pHbuffering agents. Liposomes can also be used as carriers.

A method of eliciting an immune response against a disease or pathogenin a vertebrate (e.g., vaccination) comprises administering animmunogenic composition comprising a multilayer film comprising aPlasmodium protozoan epitope. In one embodiment, the polyelectrolytecontaining the Plasmodium protozoan epitope is in the most exterior orsolvent-exposed layer of the multilayer film. The immunogeniccomposition can be administered via a wide variety of routes includingoral, intranasal, intravenous, intramuscular, subcutaneous,intraperitoneal, sublingual, intradermal, pulmonary, or transdermalroutes. The immunogenic composition can be administered in a single doseor by multiple doses spread over time to achieve optimal response andprotection. Generally, the compositions are administered in a mannercompatible with the dosage formulation, and in such amount as will beprophylactically and/or therapeutically effective. Precise amounts ofimmunogenic composition to be administered depend on the judgment of thepractitioner and may be peculiar to each subject. It will be apparent tothose of skill in the art that the therapeutically effective amount ofan immunogenic composition will depend, inter alia, upon theadministration schedule, the unit dose of antigen administered, whetherthe compositions are administered in combination with other therapeuticagents, and the immune status and health of the recipient. Atherapeutically effective dosage can be determined by the ordinaryskilled medical worker based on patient characteristics (age, weight,sex, condition, complications, other diseases, etc.), as is well knownin the art. Furthermore, as further routine studies are conducted, morespecific information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age and generalhealth of the recipient, is able to ascertain proper dosing.

The immunogenic composition optionally comprises an adjuvant. Adjuvantsin general comprise substances that boost the immune response of thehost in a non-specific manner. Selection of an adjuvant depends on thesubject to be vaccinated. Preferably, a pharmaceutically acceptableadjuvant is used. For example, a vaccine for a human should avoid oil orhydrocarbon emulsion adjuvants, including complete and incompleteFreund's adjuvant. One example of an adjuvant suitable for use withhumans is alum (alumina gel). A vaccine for an animal, however, maycontain adjuvants not appropriate for use with humans.

It is contemplated that an immune response may be elicited viapresentation of any protein or peptide capable of eliciting such aresponse. In one embodiment, the antigen is a key epitope, which givesrise to a strong immune response to a particular agent of infectiousdisease, i.e., an immunodominant epitope. If desired, more than oneantigen or epitope may be included in the immunogenic composition inorder to increase the likelihood of an immune response.

Designed peptides adsorb to the surface of an LBL films by virtue of theelectrostatic attraction between the charged surface adsorptionregions(s) of the designed peptide and the oppositely charged surface ofthe film. The efficiency of adsorption will depend largely upon thecomposition of the surface adsorption region(s). Thus designed peptideswith different epitopes but similar surface adsorption regions(s) areexpected to adsorb with similar efficiency. To fabricate a film thatcontains two distinct designed polypeptides each at a 1:1 molar ratio,one can mix the peptides at that molar ratio and deposit themsimultaneously at a particular layer. Alternatively, one could depositeach peptide individually at separate layers. The molar ratio ofpeptides adsorbed will largely mirror that relative concentrations atwhich they were layered or the number of layering steps during whichthey were incorporated.

The quantity of designed polypeptides incorporated into an LBL film canbe measured in a variety of ways. Quantitative amino acid analysis (AAA)is particularly well suited to this purpose. Films containing DP aredecomposed to their constituent amino acids by treatment withconcentrated hydrochloric acid (6 M) and heating, typically at 115° C.for 15 hours. The amounts of each amino acid are then measured usingchromatographic techniques well known to those skilled in the art. Aminoacids that occur in only one of the designed peptides in a film can beused as tracers for that peptide. When designed peptides lack uniqueamino acids, non-natural amino acids (e.g. aminobutyric acid orhomovaline) can be incorporated into designed peptides during synthesis.These tracer amino acids are readily identified during the AAAexperiment and can be used to quantitate the amount of peptide in thefilm.

As used herein, a specific T-cell response is a response that isspecific to an epitope of interest, specifically a Plasmodium protozoanepitope. A specific T-cell response is manifested by secretion of IFNγand/or IL-5 by T-cells derived from the immunized hose.

As used herein, a specific antibody response is a response that isspecific to an epitope of interest, specifically a Plasmodium protozoanepitope as disclosed herein.

As used herein, “layer” means a thickness increment, e.g., on a templatefor film formation, following an adsorption step. “Multilayer” meansmultiple (i.e., two or more) thickness increments. A “polyelectrolytemultilayer film” is a film comprising one or more thickness incrementsof polyelectrolytes. After deposition, the layers of a multilayer filmmay not remain as discrete layers. In fact, it is possible that there issignificant intermingling of species, particularly at the interfaces ofthe thickness increments. Intermingling, or absence thereof, can bemonitored by analytical techniques such as surface potentialmeasurements and X-ray photoelectron spectroscopy.

“Amino acid” means a building block of a polypeptide. As used herein,“amino acid” includes the 20 common naturally occurring L-amino acids,all other natural amino acids, all non-natural amino acids, and allamino acid mimics, for example N-alkyl glycine amino acids, oftenreferred to as peptoids.

“Naturally occurring amino acids” means glycine plus the 20 commonnaturally occurring L-amino acids, that is, alanine, valine, leucine,isoleucine, serine, threonine, cysteine, methionine, aspartic acid,asparagine, glutamic acid, glutamine, arginine, lysine, histidine,phenylalanine, ornithine, tyrosine, tryptophan, and proline.

“Non-natural amino acid” means an amino acid other than any of the 20common naturally occurring L-amino acids. A non-natural amino acid canhave either L- or D-stereochemistry.

“Peptoid,” or N-substituted glycine, means an analog of thecorresponding amino acid monomer, with the same side chain as thecorresponding amino acid but with the side chain appended to thenitrogen atom of the amino group rather than to the α-carbons of theresidue. Consequently, the chemical linkages between monomers in apolypeptoid are not peptide bonds, which can be useful for limitingproteolytic digestion.

“Amino acid sequence” and “sequence” mean a contiguous length ofpolypeptide chain that is at least two amino acid residues long.

“Residue” means an amino acid in a polymer or oligomer; it is theresidue of the amino acid monomer from which the polymer was formed.Polypeptide synthesis involves dehydration, that is, a single watermolecule is “lost” on addition of the amino acid to a polypeptide chain.

As used herein “peptide” and “polypeptide” all refer to a series ofamino acids connected one to the other by peptide bonds between thealpha-amino and alpha-carboxy groups of adjacent amino acids, and maycontain or be free of modifications such as glycosylation, side chainoxidation, or phosphorylation, provided such modifications, or lackthereof, do not destroy immunogenicity. As used herein, the term“peptide” is meant to refer to both a peptide and a polypeptide orprotein.

“Designed polypeptide” means a polypeptide that has sufficient chargefor stable binding to an oppositely charged surface, that is, apolypeptide that can be deposited into a layer of a multilayer filmwherein the driving force for film formation is electrostatics. Inspecific embodiments, a designed polypeptide is at least 15 amino acidsin length and the magnitude of the net charge per residue of thepolypeptide is greater than or equal to 0.1, 0.2, 0.3, 0.4 or 0.5 at pH7.0. In one embodiment, the ratio of the number of charged residues ofthe same polarity minus the number of residues of the opposite polarityto the total number of residues in the polypeptide is greater than orequal to 0.2 at pH 7.0. While there is no absolute upper limit on thelength of the polypeptide, in general, designed polypeptides suitablefor LBL deposition have a practical upper length limit of 1,000residues. Designed polypeptides can include sequences found in naturesuch as Plasmodium protozoan epitopes as well as regions that providefunctionality to the peptides such as charged regions also referred toherein as surface adsorption regions, which allow the designedpolypeptides to be deposited into a polypeptide multilayer film.

“Primary structure” means the contiguous linear sequence of amino acidsin a polypeptide chain, and “secondary structure” means the more or lessregular types of structure in a polypeptide chain stabilized bynon-covalent interactions, usually hydrogen bonds. Examples of secondarystructure include α-helix, β-sheet, and β-turn.

“Polypeptide multilayer film” means a film comprising one or moredesigned polypeptides as defined above. For example, a polypeptidemultilayer film comprises a first layer comprising a designedpolypeptide and a second layer comprising a polyelectrolyte having a netcharge of opposite polarity to the designed polypeptide. For example, ifthe first layer has a net positive charge, the second layer has a netnegative charge; and if the first layer has a net negative charge, thesecond layer has a net positive charge. The second layer comprisesanother designed polypeptide or another polyelectrolyte.

“Substrate” means a solid material with a suitable surface foradsorption of polyelectrolytes from aqueous solution. The surface of asubstrate can have essentially any shape, for example, planar,spherical, cubic, rod-shaped, or other shape. A substrate surface can besmooth or rough, regular or irregular. A substrate can be a crystal. Asubstrate can be a bioactive molecule. Substrates range in size from thenanoscale to the macro-scale. Moreover, a substrate optionally comprisesseveral small sub-particles. A substrate can be made of organicmaterial, inorganic material, bioactive material, or a combinationthereof. Nonlimiting examples of substrates include silicon wafers;charged colloidal particles, e.g., microparticles of CaCO₃ or ofmelamine formaldehyde; biological cells such as erythrocytes,hepatocytes, bacterial cells, or yeast cells; organic polymer lattices,e.g., polystyrene or styrene copolymer lattices; liposomes; organelles;and viruses. In one embodiment, a substrate is a medical device such asan artificial pacemaker, a cochlear implant, or a stent.

When a substrate is disintegrated or otherwise removed during or afterfilm formation, it is called “a template” (for film formation). Templateparticles can be dissolved in appropriate solvents or removed by thermaltreatment. If, for example, partially cross-linked melamine-formaldehydetemplate particles are used, the template can be disintegrated by mildchemical methods, e.g., in DMSO, or by a change in pH value. Afterdissolution of the template particles, hollow multilayer shells remainwhich are composed of alternating polyelectrolyte layers.

A “capsule” is a polyelectrolyte film in the form of a hollow shell or acoating surrounding a core. The core comprises a variety of differentencapsulants, for example, a protein, a drug, or a combination thereof.Capsules with diameters less than about 1 μm are referred to asnanocapsules. Capsules with diameters greater than about 1 μm arereferred to as microcapsules.

“Cross-linking” means the formation of a covalent bond, or severalbonds, or many bonds between two or more molecules.

“Bioactive molecule” means a molecule, macromolecule, or complex thereofhaving a biological effect. The specific biological effect can bemeasured in a suitable assay and normalizing per unit weight or permolecule of the bioactive molecule. A bioactive molecule can beencapsulated, retained behind, or encapsulated within a polyelectrolytefilm. Nonlimiting examples of a bioactive molecule are a drug, a crystalof a drug, a protein, a functional fragment of a protein, a complex ofproteins, a lipoprotein, an oligopeptide, an oligonucleotide, a nucleicacid, a ribosome, an active therapeutic agent, a phospholipid, apolysaccharide, a lipopolysaccharide. As used herein, “bioactivemolecule” further encompasses biologically active structures, such as,for example, a functional membrane fragment, a membrane structure, avirus, a pathogen, a cell, an aggregate of cells, and an organelle.Examples of a protein that can be encapsulated or retained behind apolypeptide film are hemoglobin; enzymes, such as for example glucoseoxidase, urease, lysozyme and the like; extracellular matrix proteins,for example, fibronectin, laminin, vitronectin and collagen; and anantibody. Examples of a cell that can be encapsulated or retained behinda polyelectrolyte film are a transplanted islet cell, a eukaryotic cell,a bacterial cell, a plant cell, and a yeast cell.

“Biocompatible” means causing no substantial adverse health effect uponoral ingestion, topical application, transdermal application,subcutaneous injection, intramuscular injection, inhalation,implantation, or intravenous injection. For example, biocompatible filmsinclude those that do not cause a substantial immune response when incontact with the immune system of, for example, a human being.

“Immune response” means the response of the cellular or humoral immunesystem to the presence of a substance anywhere in the body. An immuneresponse can be characterized in a number of ways, for example, by anincrease in the bloodstream of the number of antibodies that recognize acertain antigen. Antibodies are proteins secreted by B cells, and animmunogen is an entity that elicits an immune response.

“Antigen” means a foreign substance that elicits an immune response(e.g., the production of specific antibody molecules) when introducedinto the tissues of a susceptible vertebrate organism. An antigencontains one or more epitopes. The antigen may be a pure substance, amixture of substances (including cells or cell fragments). The termantigen includes a suitable antigenic determinant, auto-antigen,self-antigen, cross-reacting antigen, alloantigen, tolerogen, allergen,hapten, and immunogen, or parts thereof, and combinations thereof, andthese terms are used interchangeably. Antigens are generally of highmolecular weight and commonly are polypeptides. Antigens that elicitstrong immune responses are said to be strongly immunogenic. The site onan antigen to which a complementary antibody may specifically bind iscalled an epitope or antigenic determinant.

“Antigenic” refers to the ability of a composition to give rise toantibodies specific to the composition or to give rise to acell-mediated immune response.

As used herein, the terms “epitope” and “antigenic determinant” are usedinterchangeably and mean the structure or sequence of an antigen that isrecognized by an antibody or a T-cell. Examples of epitopes includesequences within proteins and designed polypeptides. Ordinarily anantibody epitope will be on the surface of a protein. A “continuousepitope” is one that involves several or more amino acid residues from aspan of linear peptide sequence. A “conformational epitope” involvesamino acid residues from discontinuous spans of the linear sequence of apeptide protein that are brought into spatial contact by itsthree-dimensional fold. A conformational epitope can also be comprisedof discontinuous peptide segments from distinct peptides or proteinsubunits that are brought into spatial contact by subunit quaternaryassembly. For efficient interaction to occur between the antigen and theantibody, the epitope must be readily available for binding. Thus,antibody epitopes or antigenic determinants are usually located on aproteins surface or are buried and become surface exposed by astructural rearrangement.

As used herein, a “vaccine composition” is a composition that elicits animmune response when administered to a mammal and that response protectsthe mammal against subsequent challenge by the immunizing agent or animmunologically cross-reactive agent. Protection can be complete orpartial with regard to reduction in symptoms or infection as comparedwith a non-vaccinated organism. An immunologically cross-reactive agentcan be, for example, the whole protein from which a subunit peptide hasbeen derived for use as the immunogen. Alternatively, an immunologicallycross-reactive agent can be a different protein, which is recognized inwhole or in part by antibodies elicited by the immunizing agent.

As used herein, an “immunogenic composition” is intended to encompass acomposition that elicits an immune response in an organism to which itis administered and which may or may not protect the immunized mammalagainst subsequent challenge with the immunizing agent. In oneembodiment, an immunogenic composition is a vaccine composition.

The invention is further illustrated by the following non-limitingexamples

EXAMPLES

Testing Protocols

Mice and immunizations: Female C57BL/6J, 6-8 weeks of age, were obtainedfrom Jackson Laboratories and housed at NorthEast Life Sciences, NewHaven. Mice were acclimated to the environment for at least one weekprior to use. Microparticles were resuspended in PBS to the desired DPconcentration (e.g., 10 μg/100 μl/injection) and sonicated for 10minutes immediately prior to syringe loading and immunization. Mice wereimmunized with the suspension in the rear footpad on days 0, 21 and 42.Positive control mice were immunized by subcutaneous (s.c.) injection ofDP in complete Freund's adjuvant (CFA) on d0 or DP in incompleteFreund's adjuvant (IFA) on days (d21, d42); negative control mice weremock immunized with PBS.

ELISA: Mice were bled on day 49 (post-second boost) and sera wereharvested for analysis of antibody responses using ELISA plates coatedwith T1B peptide. Antibody binding was detected with HRP-labeled goatanti-mouse IgG.

ELISPOT: Mice were sacrificed on day 49 and spleens were harvested andteased into single-cell suspensions that were depleted of erythrocytesby ammonium chloride osmotic shock. Erythrocyte-depleted spleen cellswere restimulated with T1B peptide in IFNγ or IL-5 ELISPOT plates usingcommercial reagents (BD Biosciences) and plates (Millipore Corporation)and following the manufacturers' instructions. The number of spots oneach plate was counted in an AID Viruspot Reader.

PfPb challenge: Mice were bled on day 49 and antibody titers weremeasured by ELISA as described above. Following the antibodymeasurement, mice were challenged with PfPb (Plasmodium bergheiitransgenic for the T1BT* subunit of the CS gene of P. falciparum). Thechallenge was accomplished by anesthetizing the mice and allowingPfPb-infected mosquitoes to feed on them for 10 minutes. Two dayspost-challenge, the challenged mice were bled and sacrificed, and liverRNA was extracted for analysis of parasite burden by qPCR.

Example 1: Exemplary Peptide Design and Synthesis

Designed polypeptides were based on the T1BT* multivalent peptide of P.falciparum CS. The surface adsorption region K₂₀ (SEQ ID NO: 9) or K₂₀Y(SEQ ID NO: 16) was added to the C-terminus to yield designedpolypeptides (DP) for incorporation in LbL particles (FIG. 1). WhenPam3Cys was conjugated to DP, the linker sequence SKKKK was also added.Peptides were synthesized using standard solid phase peptide chemistryprocedures and were prepared as C-terminal amides. Briefly,fluorenylmethyloxycarbonyl (Fmoc) amino acids were double coupled to aRink MBHA amide resin on a CEM Liberty microwave peptide synthesizerusing the manufacturer's synthesis protocols with minor modifications tocoupling temperatures. Following peptide synthesis, the Pam3Cys groupwas added to the resin by either manual coupling of Pam3Cys-OH orautomated coupling of Fmoc-Pam2Cys-OH followed by Fmoc removal and afinal capping step with palmitic acid. Peptides were cleaved from theresin by treatment with a trifluoroacetic acid(TFA)/triisopropylsilane/phenol/water cocktail and precipitated withether. Crude peptides were purified by C18 HPLC using a water (0.1%TFA)/acetonitrile gradient or by C4 HPLC for Pam3Cys peptides using awater (0.1% TFA)/isopropanol gradient. Purified peptides were quantifiedby UV absorbance at 280 nm or by amino acid analysis, aliquoted,lyophilized, and stored at −20° C. A typical C4 analytical HPLCchromatogram for SEQ ID NO: 13 is shown in FIG. 12 and electrospray massspectrum is shown in FIG. 13. Calculated average MW for SEQ ID NO:13=9486.27. found MW=9485.6.

T1BT*-K20: (SEQ ID NO: 10)DPNANPNVDPNANPNVNANPNANPNANPEYLNKIQNSLSTEWSPCSVTSGNGKKKKKKKKKKKKKKKKKKKKY Pam3Cys-T1BT*-K20: (SEQ ID NO: 11)Pam3CysSKKKKDPNANPNVDPNANPNVNANPNANPNANPEYLNKIQNSLSTEWSPCSVTSGNGKKKKKKKKKKKKKKKKKKKKY T1BT*-K20 (Cys->Ser):(SEQ ID NO: 12) DPNANPNVDPNANPNVNANPNANPNANPEYLNKIQNSLSTEWSPSSVTSGNGKKKKKKKKKKKKKKKKKKKK Pam3Cys-T1BT*-K20 (Cys->Ser): (SEQ ID NO: 13)Pam3CysSKKKKDPNANPNVDPNANPNVNANPNANPNANPEYLNKIQNSL STEWSPSSVTSGNGKKT1BT*-K20 (Cys->A1a): (SEQ ID NO: 14)DPNANPNVDPNANPNVNANPNANPNANPEYLNKIQNSLSTEWSPASVTSGNGKKKKKKKKKKKKKKKKKKKK Pam3Cys-T1BT*-K20 (Cys->A1a): (SEQ ID NO: 15)Pam3CysSKKKKDPNANPNVDPNANPNVNANPNANPNANPEYLNKIQNSLSTEWSPASVTSGNGKKKKKKKKKKKKKKKKKKKK T1BT*-K20 (Cys->Ser): (SEQ ID NO: 17)SKKKKDPNANPNVDPNANPNVNANPNANPNANPEYLNKIQNSLSTEWSPSSVTSGNGKKKKKKKKKKKKKKKKKKKK

Example 2: LBL Fabrication of Vaccine Microparticles

LBL was performed on a KrosFlo® Research IIi Tangential Flow FiltrationSystem from Spectrum Labs (Rancho Dominiguez, Calif.) equipped with a 20cm², 500 kD (MWCO) MicroKros® mPES filter module. Poly-L-glutamatesodium salt (PGA) and poly-L-lysine hydrobromide salt (PLL) wereobtained from Sigma-Aldrich, USA (catalog nos. P4636 and P6516,respectively). Spherical, mesoporous CaCO₃ cores (2-5 um) werecoprecipitated from 0.33 M CaCl₂ and 0.33 M Na₂CO₃ with 1.0 mg/mL PGAusing a modified version of the process reported by Volodkin et al. (D.V. Volodkin et al. Adv. Funct. Mater. 2012, 1). All steps were performedat room temperature.

The TFF apparatus was charged with 20 mL of 3% CaCO₃ (dry weight)microparticle suspension that was kept in constant circulation at 40mL/min for the duration of processing. The particles were washed bypermeation with 100 mL 10 mM HEPES buffer pH 7.4. The permeate valve wasclosed and a 5.0 mL aliquot of 6.3 mg/mL PLL was added in a singlebolus. The particles were circulated for 5 min, then the permeationvalve was opened to concentrate the suspension back to 20 mL volume. Thebuffer feed valve was opened and the particles were then washed bypermeation with 100 mL HEPES buffer. The permeate valve was closed and a5.0 mL aliquot of 5.0 mg/mL PGA was added in a single bolus. Theparticles were circulated for 5 min, concentrated, and then washed bypermeation with 100 mL HEPES buffer. The previous steps were repeateduntil a seven layer base film with PGA at the outermost layer wasfabricated.

The washed microparticle suspension was removed from the TFF apparatusand base layer film was optionally amide cross-linked (bXL=base layerscross-linked) by treatment with 200 mM EDC and 50 mM sulfo-NHS in 200 mMphosphate buffer, pH 6.5 for 30 min. The particles were pelleted by lowspeed spin, aspirated, and washed twice with 10 mM HEPES buffer toremove any residual reagent. The microparticles (either nXL or XL) werethen immersed in a 0.5 mg/mL solution of the T1BT* DP for 5 min withgentle mixing. Particles were then spun at low speed, and washed withfresh HEPES buffer to provide the final 8 peptide layer microparticlevaccine constructs. The DP loading was measured by quantitative aminoacid analysis and then the particles were suspended in HEPES buffercontaining 5% mannitol and 0.2% sodium carboxymethylcellulose. Thesuspension was aliquoted in convenient volumes (e.g. 125 ug total DP),flash frozen in liquid nitrogen, and lyophilized. The resulting drymannitol cakes were stored at 4° C. and are stable for at least sixmonths.

SEQ ID Cross- Construct NO: DP Epitope linked ACT-1198 10 ACT-2062T1BT*-K20Y N ACT-1199 11 ACT-2149 Pam3C-T1BT*-K20Y N ACT-1200 10ACT-2062 T1BT*-K20Y Y ACT-1201 11 ACT-2149 Pam3C-T1BT*-K20Y Y ACT-123615 ACT-2246 Pam3C-T1BT*-K20 N (Cys−>Ala) ACT-1237 15 ACT-2246Pam3C-T1BT*-K20 Y (Cys−>Ala) ACT-1238 13 ACT-2247 Pam3C-T1BT*-K20 N(Cys−>Ser) ACT-1239 13 ACT-2247 Pam3C-T1BT*-K20 Y (Cys−>Ser)

Example 3: Immune Phenotype Elicited by Immunization with T1BT*Microparticles

C57BL/6J mice were immunized with the indicated constructs on day 0, 21and 42. Sera collected on day 49 were tested in ELISA against T1Bpeptide as shown in FIG. 2. Results show the mean±SD of 10 mice pergroup. Sera were tested at 1:250 and plates were probed withisotype-specific detection antibodies as shown in FIG. 3. Results showmean±SD of 10 mice per group. Spleen cells were harvested on day 49 andrestimulated with T1B peptide in IFNγ and IL-5 ELISPOT plates as shownin FIG. 4. The data depict the mean±SD of 3 mice per group. nXL=nocross-linking, bXL=base layers cross-linked.

These results demonstrate that LBL microparticles loaded with designedpeptide comprising the T1BT* subunit peptide of P. falciparumcircumsporozoite protein elicit both humoral and cellular immuneresponses to the included antigenic epitopes. The results further showthat modifying the microparticles by cross-linking the base layersincreased the potency of the vaccine (higher antibody titers shown inFIG. 2). Modifying the microparticles by including a TLR2 agonistPam3Cys on the DP also increased their potency but also changed thephenotype of the immune response (increased IgG2c antibody isotype shownin FIG. 3 and decreased IL-5 response shown in FIG. 4). The antibodytiters shown in FIG. 2 further demonstrate that inclusion of bothmodifications in the same microparticle resulted in an additive benefitto the potency of the vaccine.

Example 4: Immunogenicity and Efficacy of T1BT* LbL-MP

Mice were immunized on days 0, 28, and 42 with the indicated constructs.1236 and 1237 had C→A substitution in T*, while 1238 and 1239 had C→Ssubstitution in T*. Sera were harvested on day 59 and tested in ELISAagainst T1B peptide as shown in FIG. 5. Results show the mean±SD of 10mice per group. Spleen cells were harvested on day 59 and restimulatedwith T1B peptide in IFNγ and IL-5 ELISPOT plates as shown in FIG. 6. Thedata depict the mean±SD of 3 mice per group. Mice were challenged withPfPb on day 63 and sacrificed 40 hours later as shown in FIG. 7.Parasite burden in the livers was measured by qPCR. Results show mean±SDof 10 mice per group. Insets show # of mice protected (>90% reduction in18S gene expression compared to PBS control).

These results show that all constructs elicited antibody responses toT1B peptide, but the cross-linking modification again afforded animprovement to the potency of the vaccine. Unexpectedly, ACT-1236 and-1237 (C→A substitution in T*) failed to induce IFNγ and IL-5 T-cellresponses to T1B peptide while ACT-1238 and -1239 (C→S substitution inT*) induced both T-cell responses. This difference in activity wassurprising since the substitution in these constructs is in the T*epitope which was not present in the ELISPOT assay. There is no reasonto expect the C→S substitution to retain activity while the C→Asubstitution lost activity.

Example 5: Degradation of Cysteine Containing T1BT* Peptide UnderVarious Storage Conditions

Cysteine containing T1BT* designed peptide SEQ ID NO: 10 was synthesizedas described in Example 1 and purified to >95% purity as judged by C18HPLC (FIG. 8A). Samples were dissolved in water or buffer and storedunder the following conditions: room temperature in pH approximately 5solution for 16 days (FIG. 8B), room temperature in pH 7.4 solution for2.7 days (8C), room temperature in pH 7.4 solution for 16 days (8D),storage at −20 to −10° C. as a frozen solution for 4 years (8E).Analytical HPLC was performed on these samples using a Waters X-bridgeC18 column and a gradient of 100% water/(0.075% trifluoroacetic acid(TFA)) to 50% water/acetonitrile (0.075% TFA) over 20 minutes. Thechromatograms show the original monomeric peptide at retention time 12.6minutes and the appearance of a new peak at 13.3 minutes, which is thepresumed disulfide dimer.

Example 6: Oxidation of T1BT* Designed Peptide at pH 5 and Reductionwith DTT

A 1.0 mg/mL solution of T1BT* designed peptide SEQ ID NO: 10 in waterwas prepared and the pH was estimated by pH paper to be about pH 5. Thesolution was allowed to sit at room temperature for 16 days after whicha second peak with slightly longer retention time appeared in the C18chromatogram and was presumed to be the disulfide dimer (FIG. 9A). ThepH was adjusted to 7.4 by addition of 1 M Tris buffer and a sample wasmixed 1:1 with a freshly prepared solution of 1 mg/mL dithiothreitol(DTT). After 5 minutes a second injection on HPLC showed that the latereluting peak was nearly gone, consistent with reduction back to monomerpeptide.

Example 7: Oxidation of T1BT* Designed Peptide at pH 7.4 and Reductionwith DTT

A 1.0 mg/mL solution of T1BT* designed peptide SEQ ID NO: 10 in waterwas prepared and the pH was adjusted to 7.4 by addition of 1 M Trisbuffer. The solution was allowed to sit at room temperature for 5 daysat which time most of the peptide had converted to dimer as judged bythe C18 HPLC chromatogram (FIG. 10B). The sample was then mixed 1:1 witha freshly prepared solution of 1 mg/mL DTT and after 5 min a sample wasinjected onto HPLC. The chromatogram showed nearly complete conversionback to the monomeric peptide (FIG. 10C).

Example 8: Stability of Serine Containing Designed Peptide SEQ ID NO: 17

A 1.0 mg/mL solution of T1BT* designed peptide SEQ ID NO: 17 in waterwas prepared and the pH was adjusted to 7.4 by addition of 1 M Trisbuffer. The solution was allowed to sit at room temperature for 5 daysand monitored by HPLC. The chromatogram shows the appearance of smallsatellite peaks (FIG. 11B) but most of the designed peptide remainedintact, in contrast to the cysteine containing peptide in Example 7.

The use of the terms “a” and “an” and “the” and similar referents(especially in the context of the following claims) are to be construedto cover both the singular and the plural, unless otherwise indicatedherein or clearly contradicted by context. The terms first, second,etc., as used herein are not meant to denote any particular ordering,but simply for convenience to denote a plurality of, for example,layers. The terms “comprising”, “having”, “including”, and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to”) unless otherwise noted. Recitation of ranges of valuesare merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Theendpoints of all ranges are included within the range and independentlycombinable. All methods described herein can be performed in a suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”), is intended merely to better illustrate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention as used herein.

While the invention has been described with reference to an exemplaryembodiment, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedclaims. Any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A composition comprising a multilayer filmcomprising a plurality of oppositely charged polyelectrolyte layers,wherein one of the polyelectrolyte layers in the multilayer filmcomprises a first antigenic polyelectrolyte, wherein the first antigenicpolyelectrolyte comprises a modified Plasmodium falciparumcircumsporozoite T* epitope of SEQ ID NO: 5, and wherein thepolyelectrolytes in the multilayer film comprise a polycationic materialor a polyanionic material having a molecular weight of greater than1,000 and at least 5 charges per molecule.
 2. The composition of claim1, wherein the first multilayer film is deposited on a core nanoparticleor core microparticle, or is in the form of a nanocapsule ormicrocapsule prepared by dissolving the core nanoparticle or coremicroparticle.
 3. The composition of claim 1, wherein the modifiedPlasmodium falciparum circumsporozoite T* epitope of SEQ ID NO: 5 iscovalently linked to a polycationic material or a polyanionic materialhaving a molecular weight of greater than 1,000 and at least 5 chargesper molecule.
 4. The composition of claim 1, wherein the modifiedPlasmodium falciparum circumsporozoite T* epitope of SEQ ID NO: 5 iscovalently linked to one or two surface adsorption regions at theC-terminus and/or the N-terminus of the polypeptide, wherein at leastone of the surface adsorption regions comprises five or more negativelyor positively charged amino acid residues.
 5. The composition of claim1, wherein the multilayer film further comprises a toll-like receptorligand.
 6. The composition of claim 5, wherein the toll-like receptorligand is covalently linked to the first antigenic polyelectrolyte. 7.The composition of claim 1, wherein the first antigenic polyelectrolytecomprises a modified Plasmodium falciparum circumsporozoite T1BT*epitope of SEQ ID NO:
 7. 8. The composition of claim 7, wherein themodified Plasmodium falciparum circumsporozoite T1BT* epitope of SEQ IDNO: 7 is covalently linked to a polycationic material or a polyanionicmaterial having a molecular weight of greater than 1,000 and at least 5charges per molecule.
 9. The composition of claim 7, wherein themodified Plasmodium falciparum circumsporozoite T1BT* epitope of SEQ IDNO: 7 is covalently linked to one or two surface adsorption regions atthe C-terminus and/or the N-terminus of the polypeptide, wherein atleast one of the surface adsorption regions comprises five or morenegatively or positively charged amino acid residues.
 10. Thecomposition of claim 7, wherein the first antigenic polypeptide is SEQID NO: 12 or SEQ ID NO:
 13. 11. The composition of claim 1, whereinadministration of the composition to a host organism produces anepitope-specific T-cell response, wherein the T-cell response is an IFNγT-cell response, an IL-5 T-cell response, or both.
 12. The compositionof claim 1, wherein at least two polyelectrolyte layers of themultilayer film, other than the layer containing the first antigenicpolyelectrolyte, are covalently cross-linked.
 13. The composition ofclaim 12, wherein the covalent cross-links are amide bonds involvingamino acid side chain functional groups.